During the yeast mating process, nuclear fusion occurs between two haploid nuclei of the mating pairs, forming a diploid nucleus. Therefore, membrane fusion is a critical step in the completion of zygote formation, although little is known about this process. In the simplest model, the inner and outer membranes fuse simultaneously beginning at the membranous half-bridge of the spindle pole body. In the other model, nuclear fusion actually occurs in three steps: first the outer membranes fuse, then the inner membranes fuse, and finally spindle pole body fusion completes the process. To determine whether nuclear fusion occurs in one or three steps, we are generating CFP-and YFP-tagged nuclear, ER luminal, and integral membrane proteins to follow the temporal order of their transfer during nuclear fusion. We have begun our studies using a nuclear pore marker, Nic96p, as a nuclear fusion marker. Using Nic96-CFP/-YFP, we are able to establish the timing of the nuclear fusion event in live cells and observe real-time changes in membrane shape and protein distribution during the fusion event. This information and the tools used can then be applied to other cell biological and gene regulation questions during other types of membrane fusion.